UC Davis researchers make stem cell-derived mini-brains that contain blood vessels

Growing neurons on a flat petri dish is a great way to study the inner workings of nerve signals in the brain. But I think it’s safe to argue that a two-dimensional lawn of cells doesn’t capture all the complexity of our intricate, cauliflower-shaped brains. Then again, cracking open the skulls of living patients is also not a viable path for fully understanding the molecular basis of brain disorders.

two-spheroids-in-a-dish

Brain organoids (two white balls) growing in petri dish.
Image: Pasca Lab, Stanford University.

The recent emergence of stem cell-derived mini-brains, or brain organoids, as a research tool is bridging this impasse. With induced pluripotent stem cells (iPSCs) derived from a readily-accessible skin sample from patients, it’s possible to generate three-dimensional balls of cells that mimic particular parts of the brain’s anatomy. These mini-brains have the expected type of neurons, as well as other cells that support neuron function. We’ve written many blogs, most recently in January, on the applications of this cutting-edge tool.

With any new technology, there is always room for improvement. One thing that most mini-brains lack is their own system of blood vessels, or vasculature. That’s where Dr. Ben Waldau, a vascular neurosurgeon at UC Davis Medical Center, and his lab come into the picture. Last week, their published work in NeuroReport showed that incorporating blood vessels into a brain organoid is possible.

UCDavisorganoid

A stained cross-section of a brain organoid showing that blood vessels (in red) have penetrated both the outer, more organized layers and the inner core. Image: UC Davis Institute for Regenerative Cures

Using iPSCs from one patient, the Waldau team separately generated brain organoids and blood vessels cells, also called endothelial cells. After growing each for about a month, the organoids were embedded in a gelatin containing the endothelial cells. In an excellent Wired article, writer Megan Molteni explains what happened next:

“After incubating for three weeks, they took a single organoid and transplanted it into a tiny cavity carefully carved into a mouse’s brain. Two weeks later the organoid was alive, well—and, critically, had grown capillaries that penetrated all the way to its inner layers.”

Every tissue relies on nutrients and oxygen from the blood. As Molteni suggests, being able to incorporate blood vessels and brain organoids from the same patient’s cells may make it possible to grow and study even more complex brain structures without the need of a mouse using fluidic pumps.

As Waldau explains in the Wired article, this vascularized brain organoid system also adds promise to the ultimate goal of repairing damaged brain tissue:

waldau

Ben Waldau

“The whole idea with these organoids is to one day be able to develop a brain structure the patient has lost made with the patient’s own cells. We see the injuries still there on the CT scans, but there’s nothing we can do. So many of them are left behind with permanent neural deficits—paralysis, numbness, weakness—even after surgery and physical therapy.”

 

 

Modeling the Human Brain in 3D

(Image from Pasca Lab, Stanford University)

Can you guess what the tiny white balls are in this photo? I’ll give you a hint, they represent the organ that you’re using right now to answer my question.

These are 3D brain organoids generated from human pluripotent stem cells growing in a culture dish. You can think of them as miniature models of the human brain, containing many of the brain’s various cell types, structures, and regions.

Scientists are using brain organoids to study the development of the human nervous system and also to model neurological diseases and psychiatric disorders. These structures allow scientists to dissect the inner workings of the brain – something they can’t do with living patients.

Brain-in-a-Dish

Dr. Sergiu Pasca is a professor at Stanford University who is using 3D cultures to understand human brain development. Pasca and his lab have previously published methods to make different types of brain organoids from induced pluripotent stem cells (iPSCs) that recapitulate human brain developmental events in a dish.

Sergiu Pasca, Stanford University (Image credit: Steve Fisch)

My colleague, Todd Dubnicoff, blogged about Pasca’s research last year:

“Using brain tissue grown from patient-derived iPSCs, Dr. Sergiu Pasca and his team recreated the types of nerve cell circuits that form during the late stages of pregnancy in the fetal cerebral cortex, the outer layer of the brain that is responsible for functions including memory, language and emotion. With this system, they observed irregularities in the assembly of brain circuitry that provide new insights into the cellular and molecular causes of neuropsychiatric disorders like autism.”

Pasca generated brain organoids from the iPSCs of patients with a genetic disease called Timothy Syndrome – a condition that causes heart problems and some symptoms of autism spectrum disorder in children. By comparing the nerve cell circuits in patient versus healthy brain organoids, he observed a disruption in the migration of nerve cells in the organoids derived from Timothy Syndrome iPSCs.

“We’ve never been able to recapitulate these human-brain developmental events in a dish before,” said Pasca in a press release, “the process happens in the second half of pregnancy, so viewing it live is challenging. Our method lets us see the entire movie, not just snapshots.”

The Rise of 3D Brain Cultures

Pasca’s lab is just one of many that are working with 3D brain culture technologies to study human development and disease. These technologies are rising in popularity amongst scientists because they make it possible to study human brain tissue in normal and abnormal conditions. Brain organoids have also appeared in the mainstream news as novel tools to study how epidemics like the Zika virus outbreak affect the developing fetal brain (more here and here).

While these advances are exciting and promising, the field is still in its early stages and the 3D organoid models are far from perfect at representing the complex biology of the human brain.

Pasca addresses the progress and the hurdles of 3D brain cultures in a review article titled “The rise of three-dimensional brain cultures” published this week in the journal Nature. The article, describes in detail how pluripotent stem cells can assemble into structures that represent different regions of the human brain allowing scientists to observe how cells interact within neural circuits and how these circuits are disrupted by disease.

The review goes on to compare different approaches for creating 3D brain cultures (see figure below) and their different applications. For instance, scientists are culturing organoids on microchips (brains-on-a-chip) to model the blood-brain barrier – the membrane structure that protects the brain from circulating pathogens in the blood but also makes drug delivery to brain very challenging. Brain organoids are also being used to screen for new drugs and to model complex diseases like Alzheimer’s.

Human pluripotent stem cells, adult stem cells or cancer cells  can be used to derive microfluidics-based organs-on-a-chip (top), undirected organoids (middle), and region-specific brain organoids or organ spheroids (bottom). These 3D cultures can be manipulated with CRISPR-Cas9 genome-editing technologies, transplanted into animals or used for drug screening. (Pasca, Nature)

Pasca ends the review by identifying the major hurdles facing 3D brain culture technologies. He argues that “3D cultures only approximate the appearance and architecture of neural tissue” and that the cells and structures within these organoids are not always predictable. These issues can be address over time by enforcing quality control in how these 3D cultures are made and by using new biomaterials that enable the expansion and maturation of these cultures.

Nonetheless, Pasca believes that 3D brain cultures combined with advancing technologies to study them have “the potential to give rise to novel features for studying human brain development and disease.”

He concludes the review with a cautiously optimistic outlook:

“This is an exciting new field and as with many technologies, it may follow a ‘hype’ cycle in which we overestimate its effects in the short run and underestimate its effects in the long run. A better understanding of the complexity of this platform, and bringing interdisciplinary approaches will accelerate our progress up a ‘slope of enlightenment’ and into the ‘plateau of productivity’.”

3D brain culture from the Pasca Lab, Stanford University


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Stem cell-derived mini-intestines reveal bacteria’s key role in building up a newborn’s gut

The following factoid may induce an identity crisis for some people but it is true that our bodies carry more microbes than human cells. Some studies in 1970’s estimated the ratio at 10:1 though more recent calculations suggest we’re merely half microbe, half human.

Because microbes are much smaller than human cells they make up only about 1 or 2 percent of our total body mass. But that still amounts to trillions of micro-organisms, mostly bacteria, that live on and inside our bodies. The gut is one part of our body that is teeming with bacteria. Though that may sound gross, you’re very life depends on them. For example, these bacteria allow us to digest foods and take up nutrients that we wouldn’t be able to otherwise.

Intestines

E. coli bacteria, visible in this enhanced microscope image as tiny green rods, were injected into the center of a germ-free hollow ball of cells called a human intestinal organoid (inset image, top right). Within 48 hours, the cells formed much tighter connections with one another, visible as red in this image. Image courtesy of University of Michigan.

When we’re first born our intestines are germ-free but overtime helpful bacteria gain access to our gut and help it function, protecting it from infection by the continual exposure to harmful bacteria and viruses. New research out of the University of Michigan Medical School reported in eLife now shows that the initial bacterial infiltration is even more important than scientists previously thought. It appears to play a key role in stimulating human gut cells to shore up the intestine in preparation for the full wave of both micro-organisms and pathogens that are present throughout a person’s lifetime. The finding could help researchers discover methods to protect the gut from diseases like necrotizing enterocolitis, a rare but dangerous infection that strikes newborns.

To reach these conclusions, the research team grew human embryonic stem cells into miniature intestines in the lab. These so-called human intestinal organoids, or HIOs, are structures made up of a few thousand cells that form hollow tubes with many of the hallmarks of a bona fide intestine. The HIOs were first kept in a germ-free environment to mimic a newborn’s intestine. Then a form of helpful E. Coli bacteria, the same that’s often found in an infant’s diaper, was injected into the HIO and allowed to colonize the inside of the intestine.

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A single human intestinal organoid, or HIO — a hollow ball of cells grown from human embryonic stem cells and coaxed to become gut-lining cells. Scientists can use it to study basic gut development, and the effect of microbes on the cells, in a way that mimics the guts of newborn babies. Image courtesy of University of Michigan

The team observed several changes in gene activity shortly after the bacteria was introduced. Within a day or two, genes involved in producing proteins that fight off harmful microbes increased as well as genes that encode mucus production, a key part of protecting the cells that face the inside of the intestine. Other key features of a maturing intestine, such as tighter cell-to-cell connections and lowered oxygen levels were also stimulated by the presence of the bacteria. As co-senior author Vincent Young, M.D., Ph.D. explained in a press release, these results put the team in a position to uncover new insights about intestinal biology and disease:

VBY

Vincent Young

“We have developed a system that faithfully reproduces the physiology of the immature human intestine, and will now make it possible to study a range of host-microbe interactions in the intestine to understand their functional role in health and disease.”

 

The particular mix of microbes found in one person versus another can differ a lot. And the impact of these differences on an individual’s health has been a trending topic in the media. Lead author David Hill, Ph.D., a postdoctoral fellow in the lab of Jason Spence, Ph.D., thinks that’s one specific research path that they aim to investigate with their HIO system:

HillD_spence_website

David Hill

“We hope to examine whether different bacteria produce different types of responses in the gut. This type of work might help to explain why different types of gut bacteria seem to be associated with positive or negative health outcomes.”

 

Stem cell stories that caught our eye: brains, brains and more brains!

This week we bring you three separate stories about the brain. Two are exciting new advances that use stem cells to understand the brain and the third is plain creepy.

Bioengineering better brains. Lab grown mini-brains got an upgrade thanks to a study published this week in Nature Biotechnology. Mini-brains are tiny 3D organs that harbor similar cell types and structures found in the human brain. They are made from pluripotent stem cells cultured in laboratory bioreactors that allow these cells to mature into brain tissue in the span of a month.

The brain organoid technology was first published back in 2013 by Austrian scientists Jürgen Knoblich and Madeline Lancaster. They used mini-brains to study human brain development and a model a birth defect called microcephaly, which causes abnormally small heads in babies. Mini-brains filled a void for scientists desperate for better, more relevant models of human brain development. But the technology had issues with consistency and produced organoids that varied in size, structure and cell type.

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

Fast forward four years and the same team of scientists has improved upon their original method by adding a bioengineering technique that will generate more consistent mini-brains. Instead of relying on the stem cells to organize themselves into the proper structures in the brain, the team developed a biological scaffold made of microfilaments that guides the growth and development of stem cells into organoids. They called these “engineered cerebral organoids” or enCORs for short.

In a news feature on IMBA, Jürgen Knoblich explained that enCORs are more reproducible and representative of the brain’s architecture, thus making them more effective models for neurological and neurodevelopmental disorders.

“An important hallmark of the bioengineered organoids is their increased surface to volume ratio. Because of their improved tissue architecture, enCORs can allow for the study of a broader array of neurological diseases where neuronal positioning is thought to be affected, including lissencephaly (smooth brain), epilepsy, and even autism and schizophrenia.”

Salk team finds genetic links between brain’s immune cells and neurological disorders. (Todd Dubnicoff)

Dysfunction of brain cells called microglia have been implicated in a wide range of neurologic disorders like Alzheimer’s, Parkinson’s, Huntington’s, autism and schizophrenia. But a detailed examination of these cells has proved difficult because they don’t grow well in lab dishes. And attempts to grow microglia from stem cells is hampered by the fact that the cell type hasn’t been characterized enough for researchers to know how to distinguish it from related cell types found in the blood.

By performing an extensive analysis of microglia gene activity, Salk Institute scientists have now pinpointed genetic links between these cells and neurological disease. These discoveries also demonstrate the importance of the microglia’s environment within the brain to maintain its identity. The study results were reported in Science.

Microglia are important immune cells in the brain. They are related to macrophages which are white blood cells that roam through the body via the circulatory system and gobble up damaged or dying cells as well as foreign invaders. Microglia also perform those duties in the brain and use their eating function to trim away faulty or damage nerve connections.

To study a direct source of microglia, the team worked with neurosurgeons to obtain small samples of brain tissue from patients undergoing surgery for epilepsy, a tumor or stroke. Microglia were isolated from healthy regions of brain tissue that were incidentally removed along with damaged or diseased brain tissue.

Salk and UC San Diego scientists conducted a vast survey of microglia (pictured here), revealing links to neurodegenerative diseases and psychiatric illnesses. (Image: Nicole Coufal)

A portion of the isolated microglia were immediately processed to take a snap shot of gene activity. The researchers found that hundreds of genes in the microglia had much higher activities compared to those same genes in macrophages. But when the microglia were transferred to petri dishes, gene activity in general dropped. In fact, within six hours of tissue collection, the activity of over 2000 genes in the cells had dropped significantly. This result suggests the microglial rely on signals in the brain to stimulate their gene activity and may explain why they don’t grow well once removed from that environment into lab dishes.

Of the hundreds of genes whose activity were boosted in microglia, the researchers tracked down several that were linked to several neurological disorders. Dr. Nicole Coufal summarized these results and their implications in a Salk press release:

“A really high proportion of genes linked to multiple sclerosis, Parkinson’s and schizophrenia are much more highly expressed in microglia than the rest of the brain. That suggests there’s some kind of link between microglia and the diseases.”

Future studies are needed to explain the exact nature of this link. But with these molecular descriptions of microglia gene activity now in hand, the researchers are in a better position to study microglia’s role in disease.

A stem cell trial to bring back the dead, brain-dead that is. A somewhat creepy stem cell story resurfaced in the news this week. A company called Bioquark in Philadelphia is attempting to bring brain-dead patients back to life by injecting adult stem cells into their spinal cords in combination with other treatments that include protein blend injections, electrical nerve stimulation and laser therapy. The hope is that this combination stem cell therapy will generate new neurons that can reestablish lost connections in the brain and bring it back to life.

Abstract image of a neuron. (Dom Smith/STAT)

You might wonder why the company is trying multiple different treatments simultaneously. In a conversation with STAT news, Bioquark CEO Ira Pastor explained,

“It’s our contention that there’s no single magic bullet for this, so to start with a single magic bullet makes no sense. Hence why we have to take a different approach.”

Bioquark is planning to relaunch a clinical trial testing its combination therapy in Latin America sometime this year. The company previously attempted to launch its first trial in India back in April of 2016, but it never got off the ground because it failed to get clearance from India’s Drug Controller General.

STATnews staff writer Kate Sheridan called the trial “controversial” and raised questions about how it would impact patients and their families.

“How do researchers complete trial paperwork when the person participating is, legally, dead? If the person did regain brain activity, what kind of functional abilities would he or she have? Are families getting their hopes up for an incredibly long-shot cure?”

Scientists also have questions mainly about whether this treatment will actually work or is just a shot in the dark. Adding to the uncertainty is the fact that Bioquark has no preclinical evidence that its combination treatment is effective in animal models. The STAT piece details how the treatments have been tested individually for other conditions such as stroke and coma, but not in brain-dead patients. To further complicate things, there is no consensus on how to define brain death in patients, so patient improvements observed during the trial could be unrelated to the treatment.

STAT asked expert doctors in the field whether Bioquark’s strategy was feasible. Orthopedic surgeon Dr. Ed Cooper said that there’s no way electric stimulation would work, pointing out that the technique requires a functioning brain stem which brain-dead patients don’t have. Pediatric surgeon Dr. Charles Cox, who works on a stem cell treatment for traumatic brain injury and is unrelated to Bioquark, commented, “it’s not the absolute craziest thing I’ve ever heard, but I think the probability of that working is next to zero.”

But Pastor seems immune to the skepticism and naysayers.

“I give us a pretty good chance. I just think it’s a matter of putting it all together and getting the right people and the right minds on it.”

One scientist’s quest to understand autism using stem cells

April is National Autism Awareness Month and people and organizations around the world are raising awareness about a disorder that affects more than 20 million people globally. Autism affects early brain development and causes a wide spectrum of social, mental, physical and emotional symptoms that appear during childhood. Because the symptoms and their severity can vary extremely between people, scientists now use the classification of autism spectrum disorder (ASM).

Alysson Muotri UC San Diego

In celebration of Autism Awareness Month, we’re featuring an interview with a CIRM-funded scientist who is on the forefront of autism and ASD research. Dr. Alysson Muotri is a professor at UC San Diego and his lab is interested in unlocking the secrets to brain development by using molecular tools and stem cell models.

One of his main research projects is on autism. Scientists in his lab are using induced pluripotent stem cells (iPSCs) derived from individuals with ASD to model the disease in a dish. From these stem cell models, his team is identifying genes that are associated with ASD and potential drugs that could be used to treat this disorder. Ultimately, Dr. Muotri’s goal is to pave a path for the development of personalized therapies for people with ASD.

I reached out to Dr. Muotri to ask for an update on his Autism research. His responses are below.

Q: Can you briefly summarize your lab’s work on Autism Spectrum Disorders?

AM: As a neuroscientist studying autism, I was frustrated with the lack of a good experimental model to understand autism. All the previous models (animal, postmortem brain tissues, etc.) have serious experimental limitations. The inaccessibility of the human brain has blocked the progress of research on ASD for a long time. Cellular reprogramming allows us to transform easy-access cell types (such as skin, blood, dental pulp, etc.) into brain cells or even “mini-brains” in the lab. Because we can capture the entire genome of the person, we can recapitulate early stages of neurodevelopment of that same individual. This is crucial to study neurodevelopment disorders, such as ASD, because of the strong genetic factor underlying the pathology [the cause of a disease]. By comparing “mini-brains” between an ASD and neurotypical [non-ASD] groups, we can find anatomical and functional differences that might explain the clinical symptoms.

Q: What types of tools and models are you using to study ASD?

AM: Most of my lab takes advantage of reprogramming stem cells and genome editing techniques to generate 3D organoid models of ASD. We use the stem cells to create brain organoids, also called “mini-brains” in the lab. These mini-brains will develop from single cells and grow and mature in the same way as the fetal brain. Thus, we can learn about their structure and connectivity over time.

A cross section of a cerebral organoid or mini-brain courtesy of Alysson Muotri.

This new model brings something novel to the table: the ability to experimentally test specific hypotheses in a human background.  For example, we can ask if a specific genetic variant is causal for an autistic individual. Thus, we can edit the genome of that autistic individual, fixing target mutations in these mini-brains and check if now the fixed mini-brains will develop any abnormalities seen in ASD.

The ability to combine all these recent technologies to create a human experimental model of ASD in the lab is quite new and very exciting. As with any other model, there are limitations. For example, the mini-brains don’t have all the complexity and cell types seen in the developing human embryo/fetus. We also don’t know exactly if we are giving them the right and necessary environment (nutrients, growth factors, etc.) to mature. Nonetheless, the progress in this field is taking off quickly and it is all very promising.

Two mini-brains grown in a culture dish send out cellular extensions to connect with each other. Neurons are in green and astrocytes are in pink. Image courtesy of Dr. Muotri.

Q: We’ve previously written about your lab’s work on the Tooth Fairy Project and how you identified the TRPC6 gene. Can you share updates on this project and any new insights?

AM: The Tooth Fairy Project was designed to collect dental pulp cells from ASD and control individuals in a non-invasive fashion (no need for skin biopsy or to draw blood). We used social media to connect with families and engage them in our research. It was so successful we have now hundreds of cells in the lab. We use this material to reprogram into stem cells and to sequence their DNA.

One of the first ASD participants had a mutation in one copy of the TRPC6 gene, a novel ASD gene candidate. Everybody has two copies of this gene in the genome, but because of the mutation, this autistic kid has only one functional copy. Using stem cells, we re-created cortical neurons from that individual and confirmed that this mutation inhibits the formation of excitatory synapses (connections required to propagate information).

Interestingly, while studying TRPC6, we realized that a molecule found in Saint John’s Wort, hyperforin, could stimulate the functional TRPC6. Since the individual still has one functional TRPC6 gene copy, it seemed reasonable to test if hyperforin treatment could compensate the mutation on the other copy. It did. A treatment with hyperforin for only two weeks could revert the deficits on the neurons derived from that autistic boy. More exciting is the fact that the family agreed to incorporate St. John’s Wort on his diet. We have anecdotal evidence that this actually improved his social and emotional skills.

To me, this is the first example of personalized treatment for ASD, starting with genome sequencing, detecting potential causative genetic mutations, performing cellular modeling in the lab, and moving into clinic. I believe that there are many other autistic cases where this approach could be used to find better treatments, even with off the counter medications. To me, that is the greatest insight.

Watch Dr. Muotri’s Spotlight presentation about the Tooth Fairy Project and his work on autism.

Q: Is any of the research you are currently doing in autism moving towards clinical trials?

AM: IGF-1, or insulin growth factor-1, a drug we found promising for Rett syndrome and a subgroup of idiopathic [meaning its causes are spontaneous or unknown] ASD is now in clinical trials. Moreover, we just concluded a CIRM award on a large drug screening for ASD. The data is very promising, with several candidates. We have 14 drugs in the pipeline, some are repurposed drugs (initially designed for cancer, but might work for ASD). It will require additional pre-clinical studies before we start clinical trials.

Q: What do you think the future of diagnosis and treatment will be for patients with ASD?

AM: I am a big enthusiastic fan of personalized treatments for ASD. While we continue to search for a treatment that could help a large fraction of ASD people, we also recognized that some cases might be easier than others depending on their genetic profile. The idea of using stem cells to create “brain avatars” of ASD individuals in the lab is very exciting. We are also studying the possibility of using this approach as a future diagnostic tool for ASD. I can imagine every baby having their “brain avatar” analyses done in the lab, eventually pointing out “red flags” on the ones that failed to achieve neurodevelopment milestones. If we could capture these cases, way before the autism symptoms onset, we could initiate early treatments and therapies, increasing the chances for a better prognostic and clinical trajectory. None of these would be possible without stem cell research.

Q: What other types of research is your lab doing?

Mini-brains grown in a dish in Dr. Muotri’s lab.

AM: My lab is also using these human mini-brains to test the impact of environmental factors in neurodevelopment. By exposing the mini-brains to certain agents, such as pollution particles, household chemicals, cosmetics or agrotoxic products [pesticides], we can measure the concentration that is likely to induce brain abnormalities (defects in neuronal migration, synaptogenesis, etc.). This toxicological test can complement or substitute for other commonly used analyses, such as animal models, that are not very humane or predictive of human biology. A nice example from my lab was when we used this approach to confirm the detrimental effect of the Zika virus on brain development. Not only did we show causation between the circulating Brazilian Zika virus and microcephaly [a birth defect that causes an abnormally small head], but our data also pointed towards a potential mechanism (we showed that the virus kills neural progenitor cells, reducing the thickness of the cortical layers in the brain).

You can learn more about Dr. Muotri’s research on his lab’s website.


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Brain Models Get an Upgrade: 3D Mini-Brains

Every year, companies like Apple, Microsoft and Google work tirelessly to upgrade their computer, software and smartphone technologies to satisfy growing demands for more functionality. Much like these companies, biomedical scientists work tirelessly to improve the research techniques and models they use to understand and treat human disease.

Today, I’ll be talking about a cool stem cell technology that is an upgrade of current models of neurological diseases. It involves growing stem cells in a 3D environment and turning them into miniature organs called organoids that have similar structures and functions compared to real organs. Scientists have developed techniques to create organoids for many different parts of the body including the brain, gut, lungs and kidneys. These tiny 3D models are useful for understanding how organs are formed and how viruses or genetic mutations can affect their development and ability to function.

Brain Models Get an Upgrade

Organoids are especially useful for modeling complex neurological diseases where current animal and 2D cell-based models lack the ability to fully represent the cause, nature and symptoms of a disease. The first cerebral, or brain, organoids were generated in 2013 by Dr. Madeline Lancaster in Austria. These “mini-brains” contained nerve cells and structures found in the cortex, the outermost layer of the human brain.

Since their inception, mini-brains have been studied to understand brain development, test new drugs and dissect diseases like microcephaly – a disease that causes abnormal brain development and is characterized by very small skulls. Mini-brains are still a new technology, and the question of whether these organoids are representative of real human brains in their anatomy and behavior has remained unanswered until now.

Published today in Cell Reports, scientists from the Salk Institute reported that mini-brains are more like human brains compared to 2D cell-based models where brain cells are grown in a single layer on a petri dish. To generate mini-brains, they collaborated with a European team that included the Lancaster lab. They grew human embryonic stem cells in a 3D environment with a cocktail of chemicals that prompted them to develop into brain tissue over a two-month period.

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

After generating the mini-brains, the next step was to prove that these organoids were an upgrade for modeling brain development. The teams found that the cells and structures formed in the mini-brains were more like human brain tissue at the same stage of early brain development than the 2D models.

Dr. Juergen Knoblich, co-senior author of the new paper and head of the European lab explained in a Salk News Release, “Our work demonstrates the remarkable degree to which human brain development can be recapitulated in a dish in cerebral organoids.”

Are Mini-Brains the Real Thing?

The next question the teams asked was whether mini-brains had similar functions and behaviors to real brains. To answer that question, the scientists turned to epigenetics. This is a fancy word for the study of chemical modifications that influence gene expression without altering the DNA sequence in your genome. The epigenome can be thought of as a set of chemical tags that help coordinate which genes are turned on and which are turned off in a cell. Epigenetics plays important roles in human development and in causing certain diseases.

The Salk team studied the epigenomes of cells in the mini-brains to see whether their patterns were similar to cells found in human brain tissue. Interestingly, they found that the epigenetic patterns in the 3D mini-brains were not like those of real brain tissue at the same developmental stage. Instead they shared a commonality with the 2D brain models and had random epigenetic patterns. While the reason for these results is still unknown, the authors explained that it is common for cells and tissues grown in a lab dish to have these differences.

In a Salk news release, senior author and Salk professor Dr. Joseph Ecker said that even though the current mini-brain models aren’t perfect yet, scientists can still gather valuable information from them in the meantime.

“Our findings show that cerebral organoids as a 3D model of brain function are getting closer to a real brain than 2D models, so perhaps by using the epigenetic pattern as a gauge we can get even closer.”

And while the world eagerly waits for the next release of the iPhone 7, neuroscientists will be eagerly waiting for a new and improved version of mini-brains. Hopefully the next upgrade will produce organoids that behave more like the real thing and can model complex neurological diseases, such as Alzheimer’s, where so many questions remain unanswered.

UCSF Scientists find molecular link between brain stem cells and Zika Infection

The Zika virus scare came to a head in 2015, prompting the World Health Organization to declare the outbreak a global health emergency earlier this year. From a research standpoint, much of the effort has centered on understanding whether the Zika infection is actually a cause of birth defects like microcephaly and how the virus infects mothers and their unborn children.

The Zika Virus is spread by a specific type of mosquito, the Aedes aegypti.

The Zika Virus is spread to humans by mosquitos.

What’s known so far is that the Zika virus can pass from the mother to the fetus through the placenta and it can infect the developing brain of the fetus. But how exactly the virus infects brain cells is less clear.

Brain stem cells are vulnerable to Zika

Scientists from UC San Francisco (UCSF) are tackling this question and have unraveled one more piece to the Zika infection puzzle. UCSF professor Dr. Arnold Kriegstein and his team reported yesterday in the journal Cell Stem Cell that they’ve identified a protein receptor on the surface of brain stem cells that could be the culprit for Zika virus infection.

Based on previous studies that showed that the Zika virus specifically infects brain stem cells, Kriegstein and his colleagues hypothesized that these cells expressed specific proteins that made them vulnerable to Zika infection. They looked to see which genes were turned on and off in brain stem cells derived from donated fetal tissue as well as other cell types in the developing brain to identify proteins that would mediate Zika virus entry.

AXL is to blame

They found a protein receptor called AXL that was heavily expressed in a type of brain stem cell called the radial glial cell, which gives rise to the outer layer of the brain called the cerebral cortex. AXL piqued their interest because it was identified in other studies as an entry point for Zika and other similar viruses like dengue in human skin cells. Furthermore, the team confirmed that radial glial cells produce a lot of AXL protein during development and it appears during a specific window of time – the second trimester of pregnancy.

A link between radial glial cells and Zika infection made sense to first author Tomasz Nowakowski who explained in a UCSF news release,

“In the rare cases of congenital microcephaly, these [radial glial cells] are the cells that die or differentiate prematurely, which is one of the reasons we became interested in the possible link.”

The team also found that AXL was expressed in mature brain cells including astrocytes and microglia and in retinal progenitor cells in the eye. They pointed out that the presence of AXL in the developing eye could help explain why many cases of Zika infection are associated with eye defects.

Modeling Zika infection using mini-brains

The bulk of the study used stem cells isolated from donated human fetal tissue, but the team also developed a different stem cell model to confirm their results. They generated brain organoids, also coined as “mini-brains”, in a dish from human induced pluripotent stem cells. These mini-brains contain structures and cell types that closely resemble parts of the developing brain. The team studied radial glial like cells in the mini-brains and found that they also expressed AXL on their surface.

An image of tissue that’s grown in a dish shows radial glia stem cells that are red, neurons in blue and the AXL receptor in green. Photo by Elizabeth DiLullo

Mini-brains grown in a dish have radial glia stem cells (red), neurons (blue) and the AXL receptor (green). Photo by Elizabeth DiLullo, UCSF

Kriegstein and his team believe they now have a working stem cell model for how viruses like Zika can infect the brain. Using their brain organoid model, they plan to collaborate with other UCSF researchers to learn more about how Zika infection occurs and whether it really causes birth defects.

“If we can understand how Zika may be causing birth defects,” Kriegstein said, “we can start looking for compounds to protect pregnant women who become infected.”

What’s next?

While the evidence points towards AXL as one of the major entry points for Zika infection in the developing brain, the UCSF team and other scientists still need to confirm that this receptor is to blame.

Kriegstein explained:

Arnold Kriegstein, UCSF

Arnold Kriegstein, UCSF

“While by no means a full explanation, we believe that the expression of AXL by these cell types is an important clue for how the Zika virus is able to produce such devastating cases of microcephaly, and it fits very nicely with the evidence that’s available. AXL isn’t the only receptor that’s been linked with Zika infection, so next we need to move from ‘guilt by association’ and demonstrate that blocking this specific receptor can prevent infection.”

If AXL turns out to be the culprit, scientists will have to be careful about testing drugs that block its function given that AXL is important for the proliferation of brain stem cells during development. There might be a way however that such treatments could be given to at risk women before they get pregnant.


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Scientists use cotton candy to make artificial blood vessels

Cotton candy gets a bad rap. The irresistible, brightly colored cloud of sugar is notorious for sending kids into hyperactive overdrive and wreaking havoc on teeth. While it’s most typically found at a state fair or at a sports stadium, cotton candy is now popping up at the lab bench and is re-branding itself into a useful tool that will help scientists develop artificial blood vessels for lab-grown organs.Pink_and_blue_cotton_candy

How is this sticky, sweet substance transitioning from stomachs to the lab? The answer comes from a Professor at Vanderbilt University, Dr. Leon Bellan. He develops 3D microfluidic materials for biomedical applications. Recently he and his students have tackled an obstacle that has plagued the fields of tissue engineering and 3D organ modeling – making enough blood vessels to keep engineered organs alive. The story was covered by the blog Inhabitat.

Scientists are using 3D organoids or “mini-organs” derived from stem cells to model organ development and human disease in a dish. While methods to make organoids have advanced to the point where various cell types of an organ are generated, these organoids do not develop a proper capillary system – a distribution of blood vessels that allows blood to bring water, oxygen and nutrients to tissue cells. Inevitably, cells located in the center of organoids die because they don’t have access to life-saving nutrients that the cells at the surface do.

Spinning cotton candy in the lab.

Bellan lab member spins cotton candy in the lab.

Bellan came up with a sweet solution to this problem. His team discovered that you can use cotton candy to make an artificial capillary system. Conveniently, the strands of cotton candy are similar in size to human blood vessels. Bellan and his team “spin” cotton candy fibers to generate a network of sugar strands that are held in place with a special polymer. Then, they pour a gelatinous mold over the strands, let that harden, and dissolve the sugar with an enzyme solution. What’s left is an intricate network of channels that are similar to the human capillary system.

Free of cotton candy, these artificial channels are now ready to be turned into functioning human capillaries. Bellan and his team were able to grow human endothelial cells (the cells that line your blood vessels) in these channels. The cells in these artificial blood vessels are able to survive for over a week.

cotton-candy-capillaries01-889x430

Gelatin mold with cotton candy made channels.

Their work is still preliminary but Bellan is excited about their technology’s potential for tissue engineering applications. In a video interview, he explained:

Leon Bellan. Photo by Joe Howell

Leon Bellan.
(Photo by Joe Howell)

“We’re really try to attack a fundamental hurdle for the entire field. The sci-fi version would be that you would like to be able to build an organ from scratch.”

 

Hopefully, Bellan and his group will be able to turn their sweet dream into a reality and help scientists develop properly functioning artificial organs that can be transplanted into humans.

To learn more about this fascinating technique, check out this video:


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Stem cell stories that caught our eye: watching tumors grow, faster creation of stem cells, reducing spinal cord damage, mini organs

Here are some stem cell stories that caught our eye this past week. Some are groundbreaking science, others are of personal interest to us, and still others are just fun.

Video shows tumors growing. A team at the University of Iowa used video to capture breast cancer cells recruiting normal cells to the dark side where they help tumors grow.

Led by David Soll, the team reports that cancer cells secrete a cable that can reach out and actively grab other cells. Once the cable reaches another cell, it pulls it in forming a larger tumor.

 “There’s nothing but tumorigenic cells in the bridge (between cells),” Soll said in a story in SciCasts, “and that’s the discovery. The tumorigenic cells know what they’re doing. They make tumors.”

They published their work in the American Journal of Cancer Research, and in a press release they suggested the results could provide an alternative to the theory that cancer stem cells are the engine of tumor growth.  I would guess that before too long, someone will find a way to merge the two theories into one, more cohesive story of how cancer grows.

 

3-D home creates stem cells quicker. Using a 3-D gel to grow the cells, a Swiss team reprogrammed skin cells into iPS-type stem cells in half the time that it takes in a flat petri dish. Since these induced Pluripotent Stem cells have tremendous value now in research and potentially in the future treating of patients, this major improvement in a process that has been notoriously slow and inefficient is great news.

The senior researcher Matthias Lutoff from Polytechnique Federale explained that the 3-D environment gave the cells a home closer to the environment where they would grow in someone’s body. In an article in Healthline, he described the common method used today:

 “What we currently have available is this two dimensional plastic surface that many, many stem cells really don’t like at all.”

At CIRM our goal is to get this research done as quickly as possible and to find ways to scale up any therapy so that it becomes practical to make it available to all patients who need it. Healthline quoted our CIRM scientist colleague Kevin Whittlesey on how the work would be a boon for stem cells scientists with its ability to shave months off the process of creating iPS cells.

 

Help for recent spinal cord injury.  A team at Case Western Reserve University in Cleveland used the offspring of stem cells that they are calling multi-potent adult progenitor cells (MAPCs) to modulate the immune response after spinal cord injury. They wanted to preserve some of the role of the immune system in clearing debris after an injury but prevent any overly rambunctious activity that would result in additional damage to healthy tissue and scarring.

a6353-spinalcord

They published their work in Scientific Reports and at the web portal MD the senior researcher Jerry Silver described the project as targeting a specific immune cell, the macrophage, in the early days following stroke in mice:

 

 “These were kinder, gentler macrophages. They do the job, but they pick and choose what they consume. The end result is spared tissue.”

The team injected the MAPCs into the mice one day after injury. Those cells were observed to go mostly to the spleen, which is know to be a reservoir for macrophages, and from their the MAPCs seemed to modulate the immune response.

 “There was this remarkable neuroprotection with the friendlier macrophages,” Silver explained. “The spinal cord was just bigger, healthier, with much less tissue damage.”

 

Rundown on all the mini-organs.  Regular readers of The Stem Cellar know researchers have made tremendous strides toward growing replacement organs from stem cells. You also know that with a few exceptions, like bladders and the esophagus, these are not ready for transplant into people.

Live Science web site does a fun rundown of progress with 11 different organs. They hit the more advanced esophagus and cover the early work on the reproductive tract, with items on fallopian tubes, vaginas and the penis. But most of the piece covers the early stage research that results in mini-organs, or as some have dubbed them, organoids. The author includes brain, heart, kidney, lung, stomach and liver. They also throw it the recent full ear grown on a scaffold.

Each short item comes with a photograph, mostly beautiful fluorescent microscopic images of cells forming the complex structures that become rudimentary organs.

3D printed human ear.

3D printed human ear.

Mini-stomachs.

Mini-stomachs.

This past summer we wrote about an article on work at the University of Wisconsin on the many hurdles that have to be leapt to get actual replacement organs. Progress is happening faster that most of us expected, but we still have a quite a way to go.

Four Challenges to Making the Best Stem Cell Models for Brain Diseases

Neurological diseases are complicated. A single genetic mutation causes some, while multiple genetic and environmental factors cause others. Also, within a single neurological disease, patients can experience varying symptoms and degrees of disease severity.

And you can’t just open up the brain and poke around to see what’s causing the problem in living patients. It’s also hard to predict when someone is going to get sick until it’s already too late.

To combat these obstacles, scientists are creating clinically relevant human stem cells in the lab to capture the development of brain diseases and the differences in their severity. However, how to generate the best and most useful stem cell “models” of disease is a pressing question facing the field.

Current state of stem cell models for brain diseases

Cold Spring Harbor Lab, Hillside Campus, Location: Cold Spring Harbor, New York, Architect: Centerbrook Architects

Cold Spring Harbor Lab, Hillside Campus, Location: Cold Spring Harbor, New York, Architect: Centerbrook Architects

A group of expert stem cell scientists met earlier this year at Cold Spring Harbor in New York to discuss the current state and challenges facing the development of stem cell-based models for neurological diseases. The meeting highlighted case studies of recent advances in using patient-specific human induced pluripotent stem cells (iPS cells) to model a breadth of neurological and psychiatric diseases causes and patient symptoms aren’t fully represented in existing human cell models and mouse models.

The point of the meeting was to identify what stem cell models have been developed thus far, how successful or lacking they are, and what needs to be improved to generate models that truly mimic human brain diseases. For a full summary of what was discussed, you can read a Meeting Report about the conference in Stem Cell Reports.

What needs to be done

After reading the report, it was clear that scientists need to address four major issues before the field of patient-specific stem cell modeling for brain disorders can advance to therapeutic and clinical applications.

1. Define the different states of brain cells: The authors of the report emphasized that there needs to be a consensus on defining different cell states in the brain. For instance, in this blog we frequently refer to pluripotent stem cells and neural (brain) stem cells as a single type of cell. But in reality, both pluripotent and brain stem cells have different states, which are reflected by their ability to turn into different types of cells and activate a different set of genes. The question the authors raised was what starting cell types should be used to model specific brain disorders and how do we make them from iPS cells in a reproducible and efficient fashion?

2. Make stem cell models more complex: The second point was that iPS cell-based models need to get with the times. Just like how most action-packed or animated movies come in 3D IMAX, stem cell models also need to go 3D. The brain is comprised of an integrated network of neurons and glial support cells, and this complex environment can’t be replicated on the flat surface of a petri dish.

Advances in generating organoids (which are mini organs made from iPS cells that develop similar structures and cell types to the actual organ) look promising for modeling brain disease, but the authors admit that it’s far from a perfect science. Currently, organoids are most useful for modeling brain development and diseases like microencephaly, which occurs in infants and is caused by abnormal brain development before or after birth. For more complex neurological diseases, organoid technology hasn’t progressed to the point of providing consistent or accurate modeling.

The authors concluded:

“A next step for human iPS cell-based models of brain disorders will be building neural complexity in vitro, incorporating cell types and 3D organization to achieve network- and circuit-level structures. As the level of cellular complexity increases, new dimensions of modeling will emerge, and modeling neurological diseases that have a more complex etiology will be accessible.”

3. Address current issues in stem cell modeling: The third issue mentioned was that of human mosaicism. If you think that all the cells in your body have the same genetic blue print, then you’re wrong. The authors pointed out that as many as 30% of your skin cells have differences in their DNA structure or DNA sequences. Remember that iPS cell lines are derived from a single patient skin or other cell, so the problem is that studies might need to develop multiple iPS cell lines to truly model the disease.

Additionally, some brain diseases are caused by epigenetic factors, which modify the structure of your DNA rather than the genetic sequence itself. These changes can turn genes on and off, and they are unfortunately hard to reproduce accurately when reprogramming iPS cells from patient adult cells.

4. Improve stem cell models for drug discovery: Lastly, the authors addressed the use of iPS cell-based modeling for drug discovery. Currently, different strategies are being employed by academia and industry, both with their pros and cons.

Industry is pursuing high throughput screening of large drug libraries against known disease targets using industry standard stem cell lines. In contrast, academics are pursuing candidate drug screening on a much smaller scale but using more relevant, patient specific stem cell models.

The authors point out that, “a major goal in the still nascent human stem cell field is to utilize improved cell-based assays in the service of small-molecule therapeutics discovery and virtual early-phase clinical trials.”

While in the past, the paths that academia and industry have taken to reach this goal were different, the authors predict a convergence between the paths:

“Now, research strategies are converging, and both types of researchers are moving toward human iPS cell-based screening platforms, drifting toward a hybrid model… New collaborations between academic and pharma researchers promise a future of parallel screening for both targets and phenotypes.”

Conclusions and Looking to the Future

This meeting successfully described the current landscape of iPS cell-based disease modeling for brain disorders and laid out a roadmap for advancing these stem cell models to a stage where they are more effective for understanding the mechanisms behind disease and for therapeutic screening.

I agree with the authors conclusion that:

“Moving forward, a critical application of human iPS cell-based studies will be in providing a platform for defining the cellular, molecular, and genetic mechanisms of disease risk, which will be an essential first step toward target discovery.”

My favorite points in the report were about the need for more collaboration between academia and industry and also the push for reproducibility of these iPS cell models. Ultimately, the goal is to understand what causes neurological disease, and what drugs or stem cell therapies can be used to cure them. While iPS cell models for brain diseases still have a way to go before being more clinically relevant, they will surely play a prominent role in attaining this goal.

Meeting Attendees

Meeting Attendees